Soybean [Glycine max (L.) Merr.] is one of the world's most important agronomic crops. Between 120 and 130 million acres are planted annually, resulting in 105 million tons of seed. Soybeans have dominated world oilseed production among the eight major oilseeds traded in international markets, accounting for over 97% of all world oilseed production since 1965. The value of the crop is estimated to be over 20 billion dollars. Both soybean oil and protein are used extensively in food products for human consumption. In the United States, 5% of the total protein is derived from grain legumes and up to 65% of the oil used by the food processing industry comes from soybean (Hoskin, 1987; Smith and Huyser, 1987).
Although a great deal of effort has been devoted towards the development of new cultivars of soybean with improved disease resistance, along with increased nutritional value, traditional breeding programs have been restricted because soybean germplasm is extremely narrow and the majority of the soybean cultivars in use are derived from very few parental lines (Christou et al., 1990).
Hence, modification of soybean using genetic engineering techniques would facilitate the development of new varieties with traits such as disease resistance, e.g., viral resistance, pest resistance, and herbicide resistance, and seed quality improvement in a manner unattainable by traditional breeding methods or tissue-culture induced variation. To attain genetically modified plants, a transformation system must be developed to optimize the integration of DNA in the plant, which is most commonly delivered using either an Agrobacterium-based system, which requires wounding of plant cells (Zambryski et al., 1989), or particle bombardment (Biolistics). Although transgenic soybean plants have been produced using both microprojectile bombardment (McCabe et al., 1988; Christou, P. et al., 1989) and various Agrobacterium-mediated transformation methods (Hinchee et al., 1988; Chee et al., 1989; Parrott et al., 1989; Clemente and Zhang, 2000; Di et al., 1996), legumes, including soybeans remain extremely recalcitrant to transformation (Trick, 1997). And while successes in producing transgenic plants have been reported, the frequency of attaining transgenic plants is low, e.g., Parrott et al. (1994) report 1 transgenic plant out of 195 regenerated, and Zhang et al. (1999) report that the efficiency of producing marker-positive plants in five independent attempts was 0%, 0%, 0.5%, 0.7% and 3.0%. The demand and need for new and useful transgenic soybeans is evident from the fact that transgenic soybeans, which were derived from a single transgene integration event, represent more than 50% of the total commercial production of soybeans grown in the United States. In addition, the recalcitrant nature of soybeans to transformation has rendered many molecular, genetic, and genomic techniques commonly used in other major crops, such as maize, impractical.
The “cot-node” method is a frequently used soybean transformation system based on Agrobacterium-mediated T-DNA delivery into regenerable cells in the cotyledonary node. For example, U.S. Pat. No. 5,322,783 relates to a method for transformation of soybean tissue in which cotyledonary node cells are treated with a cytokinin, and then the cells are bombarded with microparticles carrying specific vectors and exogenous DNA. U.S. Pat. Nos. 5,169,770 and 5,376,543 disclose a method in which soybean seeds are germinated, and the meristematic or mesocotyl cell tissues are inoculated with bacterial cells, specifically Agrobacterium strains which, through infection, transfer DNA into these explants.
In U.S. Pat. No. 4,992,375, a process is described in which the cotyledonary node region from a donor plant is excised, and the explant is cultured in a nutrient media containing cytokinin, until shoots arose from resultant callus. The shoots are then rooted. U.S. Pat. No. 5,416,011 also utilizes a cotyledon explant, which requires removal of the hypocotyl, saving and separating the cotyledons, and inserting a chimeric gene by inoculation with Agrobacterium tumefaciens vectors containing the desired gene. The histochemical marker GUS was employed to determine successful transformation. Nevertheless, the efficiency of the cot-node transformation system remains low apparently because of poor Agrobacterium infection of cot-node cells, inefficient selection of transgenic cells that give rise to shoot meristems, and low rates of transgenic shoot regeneration and plant establishment.
A number of reports on soybean regeneration utilized cotyledons from immature zygotic embryos induced to undergo somatic embryogenesis (Liu et al., 1992). Soybean regeneration through short meristem cultures resulted in up to 35% explants responding to plant regeneration 4 weeks after culture (Kartha et al., 1981). Regeneration via organogenesis utilizing different explants has been reported with a maximum of 97% of explants responding and 3 shoots produced per explant 10 weeks after culture, and 38% of shoots developing roots for another 4 weeks (Yeh et al., 1991). However, interactions between genotype and in vitro cultural conditions (medium, explant and light treatment) have not been reported in regeneration via organogenesis or meristem culture in soybean, although it has been studied in regeneration via somatic embryogenesis and was proven important (Powell et al., 1987; Komatsuda et al., 1991).
The unreliable transformation and regeneration of legumes in general is due, in part, to the difficulty in producing fertile mature plants from tissue culture as well as legumes being extremely resistant to Agrobacterium infection. Thus, although genes have been transferred to soybean protoplasts by electroporation of free DNA (Christou et al., 1987; Lin et al., 1987), regeneration technology for soybean has not progressed to a state where regenerated plants can be produced from protoplasts. For example, the formation of shoots, and eventual rooting, takes place only in a tiny fraction of the cases. Further, successful transformation and successful regeneration are frequently cultivar-specific, with no broad success. See, for example, Wayne et al., 1988; Finer et al., 1991; Sato et al., 1993; Moore et al., 1994; Parrott et al., 1994 and Steart et al., 1996.
Improvements have been reported in the three components of the cot-node transformation system. For example, improved selection systems and plant regeneration have been developed (Zhang et al., 1999). Considerable effort also has been applied to increasing Agrobacterium virulence by addition of chemical inducers of the vir genes (Bolton et al., 1986; Dyé et al., 1997), improvements in vir gene constructs (Hansen et al., 1994; Torisky, 1997), identification and selection of susceptible soybean cultivars (Meuer et al., 1998; Byrne et al., 1987; Delzer et al., 1990; Cho et al., 2000), and increasing the wounding of explants by either microprojectile bombardment or sonication (Bidney et al., 1992; Santarem et al., 1998).
Although agents such as dithiothreitol (DTT) and polyvinylpolypyrrolidone (PVPP) increase plant viability after Agrobacterium-mediated transformation of grape (Perl et al., 1996) and ascorbic acid, the amino acid, cysteine, and silver nitrate individually or in combination decreased damage and increased viability of Japonica rice meristem cultures and, in combination, decreased the Agrobacterium-mediated tissue necrosis of those cultures (Enríguez-Obregón et al., 1999), no agents have been reported to enhance the Agrobacterium-mediated transformation efficiency of soybeans.
Thus, what is needed is a method to reproducibly enhance the transformation of plants, e.g., soybeans.